An Intracellular Human Single Chain Antibody to Matrix Protein (M1) of H5N1 Virus

He Sun Jilin Agricultural University https://orcid.org/0000-0002-8378-3789 Guangmou Wu Chinese Academy of Agricultural Sciences Changchun Veterinary Research Institute Jiyuan Zhang Changchun University Tourism College Yu Wang Jilin Agricultural University Yue Qiu Jilin Agricultural University Hongyang Man Jilin Agricultural University Guoli Zhang Chinese Academy of Agricultural Sciences Changchun Veterinary Research Institute Zehong Li Jilin Agricultural University Yuhuan Yue (  [email protected] ) Chinese Academy of Agricultural Sciences Changchun Veterinary Research Institute Yuan Tian Chinese Academy of Agricultural Sciences Changchun Veterinary Research Institute

Research

Keywords: H5N1, HuScFv, Phage display, Antigen-binding epitopes

Posted Date: August 31st, 2021

DOI: https://doi.org/10.21203/rs.3.rs-817839/v1

License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License

Page 1/19 Abstract

Background: Infuenza virus matrix protein M1 is encoded by viral RNA fragment 7 and is the most abundant protein in virus particles. M1 is expressed in the late stages of viral replication and exerts functionality by inhibiting viral transcription. The M1 protein sequence is an attractive target for antibody drugs.

Methods: The M1 protein sequence was amplifed by RT-PCR using cDNA from the H5N1 virus as a template; the M1 protein was then expressed and purifed. A human strain, high afnity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian infuenza virus. The differences between HuScFv and TAT-HUScFv were verifed by various experiments and the amino acid binding site of the M1 protein was determined.

Results: The M1 protein of H5N1, HuScFv, and TAT-HuScFv, were successfully purifed and expressed by and in E. coli. Further analysis demonstrated that TAT-HuScFv inhibited the hemagglutination activity of the 300TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identifed as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian infuenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.

Conclusion: Recombinant HuScFv was successfully identifed using the Tomlinson (I+J) phage antibody library and successfully linked to the TAT protein transductive domain of the HIV virus. Compared with the HuScFv, the addition of the TAT peptide improved its ability to penetrate the cell membrane. A defnite amino acid binding site was identifed after the decomposition of M1 protein, thus providing a target and reference for the development of antibody drugs and the study of new drugs.

Introduction

The H5N1 virus is a highly pathogenic type A avian infuenza that can cause systemic or respiratory disease in humans (Yee, Carpenter, and Cardona 2009). However, H5N1 is mainly transmitted among birds leads to death. The highly pathogenic H5N1 avian infuenza virus has infected more than 500 million poultry worldwide since human infection with the H5N1 subtype was frst reported in Hong Kong, China, in 1997 (Chen 2009). Subsequently, human infections have emerged in Asia, Europe, and Africa, consequently leading to signifcant public health concerns (Joseph et al. 2017). The 16 sub-types of hemagglutinin HA (H1-H16) and the nine sub-types of neuraminidase NA (N1-N9) are proteins involved in avian infuenza (Noisumdaeng et al. 2021). The combination of different subtypes of HA and NA is responsible for the signifcant diversity of avian infuenza viruses.

Page 2/19 The infuenza virus matrix protein is encoded by viral RNA fragment 7; this has two open reading frames and can therefore be transcribed into two mRNAs that are translated into M1 and M2 proteins, respectively (Raman et al. 2020). The M1 protein, with molecular weight of approximately 26 kD, is found extensively in virions, and its sequence is highly conserved. Based on these characteristics, M1 has been used as the basis for classifying infuenza viruses into types A, B and C. M1 protein is expressed in the late stages of viral replication and is concentrated in discrete cell lacunae (Poungpair et al. 2009). A small amount of M1 protein translocates to the nucleus during the late stages of viral infection and helps to inhibit viral transcription. Thereafter, M1 interacts with the NEP (NS2 protein) tail encoded by viral RNA fragment 8 to export vRNP from the nucleus to the cytoplasm (Mok et al. 2017; Paterson and Fodor 2012), thus triggering the budding process and the release of virions. M1 and RNA fragments that encode M1 are the most attractive target sites for antibody drugs, gene silencing, and drug therapy (Dong-Din-On et al. 2015; Poungpair et al. 2010).

In this study, a single chain antibody with high afnity was screened by the application of a phage antibody library-Tomlinson I + J based on the M1 protein of the H5N1 virus as a target. In view of the transduction properties of protein transduction domain (PTD)-mediated protein across the cell membrane, the PTD of HIV was used to link to the single-chain antibody molecules (Yin et al. 2020; Shim et al. 2018; Lin and Kao 2015). Following the expression of this segment as a fusion protein with HuScFv, we observed efcient the efcient transfer of TAT-HuScFv into virus-infected cells (Zhang, Zhang, and Wang 2012; Theisen et al. 2006) and the specifc binding of the intracellular M1 protein, thus preventing the assembly and release of the infuenza virus and suggesting that this antibody could be used as an antiviral drug.

Materials And Methods

Vectors, Bacterial Strains, Helper Phage, and Libraries

The cloning and expression vector pET-Sumo, E. coli strain BL21 (DE3) and SUMO enzyme were purchased from Invitrogen Biotechnology Co. (CA, USA). The pET28a-TAT-GFP vector was constructed previously by our research group. A Tomlinson (I+J) phage antibody library, E. coli strains (TG, HB2151) and helper phage (KM13) were obtained from the Medical Research Council in Cambridge, UK. Goat anti- mouse IgG labeled horseradish peroxidase (HRP) antibody and anti-HIS6 labeled antibody were purchased from Abbot Trading Co., Ltd (Shanghai, China). The Protein A-HPR antibody was purchased from Abcam Co. (Cambridge, England). SP Sepharose 4 FF, Chelated Sepharose 4 FF, rProtein-A Sepharose 4 FF, and AKTA Prime chromatographs, were purchased from GE Healthcare (Fairfeld, CT, USA). Stocks of H5N1 (A/Meerkat/Shanghai/SH-1/2012) and H1N1 (A/Changchun/01/2009) viruses are held in our Institute.

Expression and Purifcation of H5N1-M1 Protein

The cDNA sequence of the M1 protein for H5N1 virus was identifed in the NCBI library; this sequence was then used as a template for primer design: forward: 5′-ATG AGT CTT CTA ACC GAG GTC-3′ and reverse: 5′- Page 3/19 CCG GAA TTC TTA CTT GAA TCG CTG CAT CTG CAC T-3′. The M1 gene was amplifed using H5N1 (A/Meerkat/Shanghai/SH-1/2012) cDNA as a template. The PCR reaction conditions were as follows: 94°C denaturation for 5 min, 94°C for 50 s, 50°C for 50 s, 72°C for 50 s (for 34 cycles) followed by 72°C 10 min. The amplifcation products were separated by gel electrophoresis and then ligated into the PET- SUMO vector with T4 ligase. The vector was sequenced and then transformed into BL21 (DE3) competent cells.

Biopanning of a Phage Display Library and Selection by M1 specifcity

The phage antibody library was prepared in accordance with the manufacturer's instructions and then screened with the purifed M1 protein as an antigen. During this process, the M1 protein was coated in 96- well plates (Nunc-Nalgene, USA) at a concentration of 5µg/ well and then incubated at 4°C overnight. The next day, the supernatant was discarded and washed three times with PBS to remove the non-adsorbed antigen. Non-specifc binding was blocked with 200 µL of 2% milk/PBS at 37°C for 2 hours. After discarding the sealing solution, the wells were washed three times with PBS, and the liquid was removed by vigorous shaking. Next, the Tomlison I+J phage antibody library was added and diluted with 2% milk/PBS to a titer of 1.0 ×1013; 100 µL was added to each well, and the liquid was incubated with vigorous shaking at room temperature for 60 min. After standing for 60 min, the liquid was discarded, and the wells were washed 10 times with PBS (0.05% (V/V) Tween-20). The residual liquid in each well was patted dry and 50µL of eluting solution (5 mg/mL trypsin-PBS) was added to each well. The plates were then shaken at room temperature for 15 min to eluate the phages, which were then stored at 4°C. The eluded phage was cloned into E. coli TG1 and further panning was performed. Second, third, and fourth rounds of panning were performed under similar conditions, except that the concentration of the antigen coating was reduced to 2 µg/well. Unbound phages were removed by 20, 30, and 40 washes with PBS (0.05% (V/V) Tween-20).

Next, 2% milk/PBS (100µL/Well) was added to the plate, and the plate was kept overnight at 4℃. Nonspecifc binding was then blocked with 2% BSA/PBS for 2 h and the phage antibody from the fourth round of screening was added. After incubation at room temperature for 1 h, the supernatant was collected to remove the phages that had been specifcally adsorbed to the milk powder in the antibody library; the collected phages were then stored at 4°C.

Expression of Positive Clones and ELISA Analysis for M1 Protein

After four rounds of screening, 10µL of phages were added to 200µL of fresh E. coli HB 2151 and left for 30 min in a water bath at 37°C. Then, 50 µL was applied to a TYE (15 g bacto-agar, 8 g Nacl, 10 g tryptone, 100g Ampicillin, 10g Glucose, 5 g yeast extract in 1L) plate and cultured overnight at 37°C. Once grown on the plate, single colonies were randomly selected and placed on a 96-well culture plate; each well contained 100 µL of 2×TY (30 g bacto-agar, 16 g Nacl, 20 g tryptone, 100g Ampicillin, 10g Glucose, 10 g yeast extract in 1L) medium and cultured overnight at 37°C. The next day, approximately 2 µL of bacterial solution from each well was placed in another 96-well cell plate (the remaining solution was

Page 4/19 added to glycerol at a fnal concentration of 15% and stored at -70°C). The new cell plate contained 200µL of 2×TY medium (containing 100 µg/mL Amp and 0.1% glucose) from each well and cultured at

37°C to an OD600 of 0.9 (after approximately 4 h of culture). Isopropyl β-D-Thiogalactoside (IPTG) at a fnal concentration of 1 mmol/L was added to each well and cultured overnight on a 30°C shaker. After overnight culture, the bacterial solution was centrifuged at 1,800 g for 15 min; the supernatant was then transferred to a new plate and stored at 4°C to await testing.

M1 protein (2 µg, 100 µL/well) was added to 96-well plates and incubated overnight at 4°C. The next day, the plates were washed three times (3 min each time) with wash solution (0.05% PBS (V/V) and Tween- 20). Next, 200 µL of 2% milk/PBS was added to each well and incubated at 37°C for 1 h. Then, 100 µL of HB2151-induced supernatant was used as a negative control; this was added to each well and incubated at 37°C for 1h. Next, the enzyme label plate was washed three times (for 3min each time) and the excess liquid was patted dry. Next, 100 µL (1:500) of Protein A-HRP was added to each well and incubated at 37°C for 1h. Washing was carried out three more times (3min each time) and excess liquid was patted dry. o-Phenylenediamine (OPD) solution (100 µL) was then added to each well and incubated at room temperature in the light for 20 min. Finally, 2 mol/L of sulfuric acid (50 µL) was added to each well to stop the reaction and the OD490 absorption value was determined.

Sequence Determination of Selected Phage Clones and the Expression & Purifcation of HuScFv

The M1-positive binding phage in the monoclonal ELISA was used as a template, and vector specifc primers (LMB3: 5ʹ-CAG GAA ACA GCT ATG AC-3ʹ; PHEN: 5ʹ-CTA TGC GGC CCC ATT CA-3ʹ) were used to amplify the HuScFv gene fragment. The obtained amplifcation products were subsequently detected by 1% agarose gel electrophoresis. The PCR amplifcation conditions were as follows: pre-denaturation at 94°C for 5 min, 94°C for 50 s, 54°C for 50 s, 72°C for 120 s (35 cycles) and a fnal extension at 72°C for 10 min. The target DNA fragment was then recovered and sequenced by Kumei Biological Engineering Co. (China).

ELISA-positive strains were transferred to 5 mL of 2× TY medium containing 100 µg/mL Amp and 1% glucose and cultured overnight at 37°C. The next day, 200 µL of overnight culture was transferred to 2×

TY medium (containing 100 µg/mL Amp and 0.1% glucose) and cultured at 37°C to an OD600 of 0.9 (approximately 4 h). A fnal concentration of 1 mmol/L of IPTG was added for overnight induction on a shaking table at a 30°C. On the third day, the induced bacterial solution was centrifuged at 5000 g (Beckman, USA) for 30 min; the supernatant was removed and precipitated with 10%–55% saturated ammonium sulfate. The precipitated solution was then resuspended with 30 mmol/L of PB (pH7.2) and dialysis was performed in PBS overnight. The crude samples were then purifed by Protein-A FF afnity chromatography; eluted samples were dialyzed with PBS overnight. The target protein was fnally analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Western Blotting & Immunoafnity Analysis with HuScFv

Page 5/19 The purifed M1 protein was separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane using a protein electrophoresis transfer device (BIO-RAD) at 45 V for 35min. Membranes were then blotted with HuScFv as a primary antibody and ProteinA-HRP as a secondary antibody (diluted with 1:2,000 PBS). DAB was used as a color reagent to detect the immunobinding activity of the antibody. The binding ability of HuScFv to M1 was determined by a non-competitive ELISA method. Different concentrations of the M1 protein antigen were coated with skimmed milk powder for 1 h, and different dilutions of HuScFv were added. Then, proteinA-HRP and OPD color solution were added; 2 M of sulfuric acid was used as a termination solution, and the absorbance was measured at 490 nm wavelength. Afnity constants were calculated using the formula Ka . IgBLAST (a tool for immunoglobulin (IG) and T cell receptor (TR) V domain sequences, NCBI) was used to analyze the nucleotide sequences of clones with the highest immune afnity constant to determine the HuScFv framework and complementary determination region (CDR).

Expression and Purifcation of TAT-HuScFv

Forward (5'-GTG AAT TCA TAA TGA AAT ACC TAT TGC CT-3') and reverse (5 '-GCA AGC TTC TAT GCG GCC CCA TTC AG-3') sequences were introduced into EcoR I and Hind III sites, respectively. A HuScFv plasmid with a high immunoafnity constant was extracted and used as a template for PCR. The PCR reaction conditions were as follows: pre-denaturation at 94°C for 5 min, then 34 cycles of 94°C for 50 s, 50°C for 50 s, and 72℃; this was followed by a fnal extension at 72°C for 10 min and cooling at 4°C. Amplifcation products were recovered by gel electrophoresis. The amplifcation products and the vector (pET28a-TAT-GFP) were extracted by the double digestion of EcoR I and Hind III, respectively; the amplifcation product was then ligated with T4 ligase and transformed into competent E. coli DH5α. Positive clones were then identifed by PCR.

The pET28A-TAT-HuScFv construct was then transformed into BL21(DE3) with the CaCl2 method. A single colony was selected and inoculated into LB liquid medium (containing 50 μg/mL Kan) at 37°C and

180 rpm until the OD600 was approximately 0.5. IPTG was added to a fnal concentration of 1 mmol/L and induction was carried out over a culture period of 4 h. The induced bacterial culture was centrifuged at 4°C at 5000g for 20 min, and the bacteria were collected. The bacteria were suspended with TE (pH 8.0) buffer solution and ultrasonically crushed in an ice bath (power: 1500 W; working time: 5 s; interval time: 9 s; 50 times in total). Microscopic examination confrmed that the bacteria had been completely broken down. Centrifugation was carried out at 12,000 g; samples of supernatant and precipitate were then separated by 12% SDS-PAGE.

The supernatant of the expressed cells was obtained following ultrasonic lysis, and a metal chelated Cu2+ column was used as the buffer system for PBS (pH 7.2). The elution peaks of the target proteins were analyzed with 20 mM and 200 mM imidazole, respectively. The eluent was then purifed on a rProteinA FF column. Finally, eluted samples were dialyzed with PBS.

Hemagglutination Inhibition Analysis of Anti-M1-HuScFv and TAT-HuScFv

Page 6/19 Digested MDCK cells were placed in a 96-well cell culture plate (3×104 cells/well), and the cells grown into a single layer to be absorbed into the medium. The cells were washed three times with DMEM, and A/Changchun/01/2009 H1N1 was added to each well at different concentrations (PBS was added to the negative control well). The cells were incubated at 37°C for 3.5 h, and the extracellular fuid was discarded. Cells were washed twice with PBS; purifed HuScFv and TAT-HuScFv (10 μg/well; the positive control included PBS only) were then added and incubated at 37°C for 1.5 h. The supernatant of cells was then absorbed and DMEM (containing 1% FBS) was added to each well and cultured overnight at

37°C with 5% CO2. The next day, the hemagglutination inhibition test was performed with the overnight culture supernatant. The supernatant was added to the reaction plate (50 μL/well), along with fresh 0.85% chicken red blood cell suspension (50 μL/well), and incubated at room temperature for 30 min; the results of the test were observed by the upright reaction plate.

TAT-HuScFv Bound to Amino Acid Sites of M1 Protein Epitopes

The M1 protein was sequenced and then decomposed in order from the N-terminal to the C-terminal to synthesize 10 polypeptides (Table 1). Positive fragments were detected by the sandwich ELISA method, as described earlier. The polypeptides were used to coat 96-well plates, using TAT-HuScFv as the frst antibody, and protein A-HRP as the second antibody. Analysis involved 3, 3′,5 ,5′-Tetramethylbenzidine (TMB) color and the positive fragments. The positive fragments were decomposed into peptides according to the overlapping sequences of four amino acids and then detected by sandwich ELISA. Positive results were analyzed and positive fragments were synthesized into peptides; these were tested again by sandwich ELISA until the amino acid sites that bound to TAT-HuScFv were identifed.

Results Preparation of the H5N1-M1 Protein

The M1 protein (H5N1, A/Meerkat/Shanghai/SH-1/2012) gene was successfully amplifed and cloned into the pET-Sumo-TAT vector. PCR identifcation revealed a target band at 750 bp (Fig. 1A). Sequencing results showed that the sequence encoding the M1 protein had been cloned into the vector in the correct reading frame. The expression vector was transformed into E. coli BL21 (DE3) and induced with IPTG (at a fnal concentration of l mmol/l). The target protein (purity>95%) (Fig. 1B) was successfully obtained and was 30KDa in size.

The Use of a Phage Display Library to Identify HuScFv Specifc to M1 H5N1

Using purifed M1 protein as an antigen and the Tomlinson (I + J) phage antibody library, we identifed four strains of anti-M1 protein HuScFv by four rounds of biopanning (1C, 7B, 3F and 4G). The PCR

Page 7/19 method was used to determine whether the positive strains with biological activity had been detected successfully and specifc fragments of 930 bp were amplifed from the four positive strains (Fig. 2A).

Western blotting was performed for the ELISA-positive strains and M1 protein. Analysis showed that 3F and 7B had strong binding ability with the M1 protein and that the stain location and stain depth were more advantageous than other forms of HuScFv under the same conditions (Fig. 2B). Afnity constants for 1C, 7B, 3F and 4G were determined by uncompetitive enzyme immunoassay, and a total of four Ka values were obtained according to the simulated biological curve of protein concentration for M1 (Table 2). IgBLAST was used to analyze 3F in the NCBI library to determine the HuScFv framework and CDR region (Fig. 3).

Characterization Of The Recombinant Tat-huscfv Trans-body For M1

The 3F and 7B genes (with the highest immune afnity constant) were successfully cloned into the pET28a-TAT expression vector (Fig. 4) and successfully expressed into BL21 (DE3). SDS-PAGE further showed that the protein product was approximately 28KDa in size (Fig. 5); this was the expected size of the fusion protein. TAT-HuScFv was highly expressed in IPTG-induced E. coli, and was eluted in 200 mmol of imidazole when purifed by the metal chelated Cu2+ column. The purifed TAT-HuScFv and HuScFv were compared for hemagglutination inhibition; the ability of TAT-HuScFv when fused to the TAT domain to bind viral M1 protein was stronger than that of HuScFv (Table 3).

Specifc Recognition Sites Of M1 Protein For Tat-huscfv

ELISA was conducted between purifed TAT-HuScFv and small peptides based on M1-protein decomposition. Only fragment 10 (Table 1) was positive; all nine of the other fragments were negative. Because polypeptide number 10 had only 26 amino acid sequences; four of its amino acids were overlapped to synthesize multiple small peptides. After ELISA was conducted again, peptides 5, 6 and 7 were all positive (Fig. 6A). These three small peptides all contained the same two amino acids (LENLQA, NLQAYQ, QAYQKR, QA). Two polypeptides, containing eight amino acids (Q A) were synthesized; then, Q A was genetic mutated into E (ENLEAYQK) G(ENLEQGYQK) respectively. Finally, ELISA (the disordered peptide with the same sequence as the source peptide was also the negative control) was performed (Fig. 6B); peptide 1 was positive and peptide 2 was negative. Therefore, it was inferred that TAT-HuScFv specifcally bound to Alanine (A) at position 239 in the M1 protein.

Page 8/19 Table 1 Polypeptide sequence of the M1 protein Number Peptide sequence based on M1 protein

1 N-MSLLTEVETYVLSIIPSGPLKAEIA-C

2 N-QKLEDVFAGKNTDLEALMEWLKTRP-C

3 N-ILSPLTKGILGFVFTLTVPSERGLQ-C

4 N-RRRFVQNALNGNGDPNNMDRAVKLY-C

5 N-KKLKREITFHGAKEVALSYSTGALA-C

6 N-SCMGLIYNRMGTVTTEVAFGLVCAT-C

7 N-CEQIADSQHRSHRQMATITNPLIRH-C

8 N-ENRMVLASTTAKAMEQMAGSSEQAA-C

9 N-EAMEVANQARQMVQAMRTIGTHPNS-C

10 N-SAGLRDNLLENLQAYQKRMGVQMQRF-C

Table 2 The afnity constants were measured as a Ka value HuScFv n = 2 n = 4 n = 8 ±s

1C(×106) 2.7 3.42 2.95 3.15 3.09 3.03 3.06 ± 0.22

7B(×107) 1.26 2.4 4.67 1.85 8.02 4.55 3.79 ± 2.29

3F(×108) 2.8 2.44 3.95 2.55 3.27 3.2 3.04 ± 0.51

4G(×107) 3.27 3.97 2.04 3.71 2.44 2.53 2.99 ± 0.70

Page 9/19 Table 3 Hemagglutination inhibition test results Antibody H1N1*(TCID50)

50 100 150 200 250 300 350 400

3F - - - - + + + +

TAT-3F ------+ +

7B - - - - + + + +

TAT-7B - - - - - + + +

Positive control + + + + + + + +

Negative control ------

*The TCID50 of H1N1 was 10 − 4.5/0.1mL, positive(+), negative(-).

Discussion

New resistant strains of infuenza virus are emerging all over the world and the limitations associated with existing treatment drugs for infuenza virus are becoming increasingly apparent (Uyeki 2009), Consequently, there is an urgent need to develop remedies for infuenza. While traditional small molecule drugs are not suitable for inhibiting the protein-protein interface (PPI), we developed a fully human small antibody fragment (human HuScFv) for the treatment of infuenza (C. Li, Bu, and Chen 2014; Alizadeh et al. 2020). The HuScFv fragment is safe for use in different populations because it is human in origin and does not cause additional infammation. Typically, each specifc antibody molecule binds to its target using several amino acid residues in the complementary determination region (CDR) and the immunoglobulin framework (FR) of the VH and VL domains. Antibody drugs have an advantage over traditional small-molecule drugs in terms of responding to viral mutations (Ascione et al. 2009). Over recent years, human antibodies based on HA protein encoded by infuenza virus fragment 4, non- structural protein NS1 encoded by infuenza virus fragment 8, and M2 protein encoded by infuenza virus fragment 7, have been reported to inhibit viral activity, viral replication, and viral ion channel proteins, respectively (Terrier et al. 2012; Pissawong et al. 2013; Wu et al. 2014; T. W. Li et al. 2016; Poursiyami, Nejatollahi, and Moatari 2019). In addition, it has been reported that the M1 protein was highly homologous and conserved in mutant strains of infuenza A (Poungpair et al. 2010; Dong-Din-On et al. 2015). Consequently, specifc antibodies against M1 protein will exhibit the key advantages of universality and versatility.

In the present study, we used a Tomlinson (I + J) phage antibody library to isolate a unique HuScFv that exhibited high afnity for the infuenza virus M1 protein. By sequencing the HuScFv gene, we were able to fuse an HIV TAT protein transduction domain to the HuScFv fragment. Using MDCK cells, we demonstrated that TAT-HuScFv penetrated the cell membrane rapidly, entered cells, and specifcally

Page 10/19 bound with M1 protein. We identifed that the binding site was the Alanine (A) at position 239 in the M1 protein. When used to treat infuenza virus infection, the penetrating TAT-HuScFv could signifcantly shorten the treatment time and improve the efciency of treatment. In conclusion, the penetrating TAT- HuScFv generated in this study could substantially neutralize a large number of viruses in mammalian cells, and exerted signifcant effects on the life cycles of viruses. Therefore, this study provided a solid foundation for the protection and treatment of avian infuenza virus H5N1 and its related subtypes.

Conclusions

We developed a recombinant HuScFv antibody with high afnity for infuenza virus M1 protein by using the Tomlinson (I + J) phage antibody library. TAT-HuScfv was further enhanced by binding to HuScFv fragment via the TAT protein transductive domain of the HIV virus. The binding site was identifed as alanine (A) at the 239th position of M1 protein.

Abbreviations

PTD: protein transduction domain; BSA: Bovine serum albumin; IPTG: Isopropyl β-D-Thiogalactoside; OPD: o-Phenylenediamine solution; ELISA: Enzyme linked immunosorbent assay

SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; CDR: Complementary determination region; FBS: Fetal bovine serum; TMB: 3, 3′,5 ,5′-Tetramethylbenzidine; FR: Framework; AIDS: Acquired immunodefciency syndrome; WB: Western Blotting.

Declarations

Acknowledgements

The authors would like to thank Dr. Guoli Zhang for data analysis and the support of the Changchun Veterinary Research Institute, Chinese Academy of Agricultural Sciences. We would also like to thank International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript.

Author contributions

HS was responsible for analysis, investigation; writing, reviewing, and editing; and wrote the frst draft of the manuscript. GW and JZ were responsible for validation. YW and HM were responsible for software. YQ contributed to the investigation. GZ supervised the project. ZL was responsible for visualization. YY and YT were responsible for validation. All authors read and approved the fnal manuscript.

Funding

This research was supported by the Fundamental Research Funds for Science and Technology Department of Jilin Province (Reference: 20200404113YY).

Page 11/19 Availability of data and materials

The datasets analyzed and generated during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Conficts of Interests

The authors have no conficts of interest to declare.

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Page 12/19 Virus Targeting Hemagglutinin Subunit 2 (HA2).” Archives of Virology 161 (1): 19–31. https://doi.org/10.1007/s00705-015-2625-6. 8. Lin, Bo Yu, and Mou Chieh Kao. 2015. “Therapeutic Applications of the TAT-Mediated Protein Transduction System for Complex I Defciency and Other Mitochondrial Diseases.” Annals of the New York Academy of Sciences 1350 (1): 17–28. https://doi.org/10.1111/nyas.12858. 9. Mok, Bobo Wing Yee, Honglian Liu, Pin Chen, Siwen Liu, Siu Ying Lau, Xiaofeng Huang, Yen Chin Liu, Pui Wang, Kwok Yung Yuen, and Honglin Chen. 2017. “The Role of Nuclear NS1 Protein in Highly Pathogenic H5N1 Infuenza Viruses.” Microbes and Infection 19 (12): 587–96. https://doi.org/10.1016/j.micinf.2017.08.011. 10. Noisumdaeng, Pirom, Thaneeya Roytrakul, Jarunee Prasertsopon, Phisanu Pooruk, Hatairat Lerdsamran, Susan Assanasen, Rungrueng Kitphati, Prasert Auewarakul, and Pilaipan Puthavathana. 2021. “T Cell Mediated Immunity against Infuenza H5N1 Nucleoprotein, Matrix and Hemagglutinin Derived Epitopes in H5N1 Survivors and Non-H5N1 Subjects.” PeerJ 9: 1–21. https://doi.org/10.7717/peerj.11021. 11. Paterson, Duncan, and Ervin Fodor. 2012. “Emerging Roles for the Infuenza A Virus Nuclear Export Protein (NEP).” PLoS Pathogens 8 (12). https://doi.org/10.1371/journal.ppat.1003019. 12. Pissawong, Tippawan, Santi Maneewatch, Kanyarat Thueng-In, Potjanee Srimanote, Fonthip Dong- Din-On, Jeeraphong Thanongsaksrikul, Thaweesak Songserm, Pongsri Tongtawe, Kunan Bangphoomi, and Wanpen Chaicumpa. 2013. “Human Monoclonal ScFv That Bind to Different Functional Domains of M2 and Inhibit H5N1 Infuenza Virus Replication.” Virology Journal 10 (1): 1. https://doi.org/10.1186/1743-422X-10-148. 13. Poungpair, Ornnuthchar, Wanpen Chaicumpa, Kasem Kulkeaw, Santi Maneewatch, Kanyarat Thueng- in, Potjanee Srimanote, Pongsri Tongtawe, Thaweesak Songserm, Porntippa Lekcharoensuk, and Pramuan Tapchaisri. 2009. “Human Single Chain Monoclonal Antibody That Recognizes Matrix Protein of Heterologous Infuenza A Virus Subtypes.” Journal of Virological Methods 159 (1): 105– 11. https://doi.org/10.1016/j.jviromet.2009.03.010. 14. Poungpair, Ornnuthchar, Anek Pootong, Santi Maneewatch, Potjanee Srimanote, Pongsri Tongtawe, Thaweesak Songserm, Pramuan Tapchaisri, and Wanpen Chaicumpa. 2010. “A Human Single Chain Transbody Specifc to Matrix Protein (M1) Interferes with the Replication of Infuenza a Virus.” Bioconjugate Chemistry 21 (7): 1134–41. https://doi.org/10.1021/bc900251u. 15. Poursiyami, Mahboubeh, Foroogh Nejatollahi, and Afagh Moatari. 2019. “Isolation of Neutralizing Human Single Chain Antibodies against Conserved Hemagglutinin Epitopes of Infuenza a Virus H3N2 Strain.” Reports of Biochemistry and Molecular Biology 8 (3): 301–9. 16. Raman, Hadia Syahirah Abd, Swan Tan, Joseph Thomas August, and Asif M. Khan. 2020. “Dynamics of Infuenza A (H5N1) Virus Protein Sequence Diversity.” PeerJ 2020 (5). https://doi.org/10.7717/peerj.7954. 17. Shim, Byoung Shik, In Su Cheon, Eugene Lee, Sung Moo Park, Youngjoo Choi, Dae Im Jung, Eunji Yang, et al. 2018. “Development of Safe and Non-Self-Immunogenic Mucosal Adjuvant by

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Figures

Page 14/19 Figure 1

Electrophoretic images showing gene amplifcation and PCR identifcation of the recombinant plasmid (A) and the purifed M1 protein (B). (A) Lane M: DL2000; lane 1: the M1 gene was amplifed with primers M1P1 and M1P2 using the H5N1 cDNA as a template; lanes 2 and 3: using the plasmid as a template, M1P1 and M1P2 amplifcation; lane 4, negative control; (B) Lane M: protein maker, lane 1: purifed M1 protein.

Figure 2

Page 15/19 Results of PCR amplifcation and ELISA positive strains (A) and western blotting results for 3F and 7B (B). (A) Lane M: DL2000 DNA Maker. Lanes 1 and 4: specifc fragments of ELISA-positive strains. (B) Under the same conditions, 3F and 7B exhibited signifcant advantages over other proteins.

Figure 3

DNA sequence and deduced amino acid sequence. The orange sections show the variable regions CDR1, CDR2, and CDR3 of the heavy chain. The blue section is the protein peptide linker. The green section shows the variable regions CDR1, CDR2, and CDR3 of the light chain; the non-colored section is the frame area.

Page 16/19 Figure 4

PCR identifcation of the recombinant expression plasmid pET28a-TAT-HuScFv. Lane M: DL2000 DNA marker; lanes 1 and 4: PCR product for the recombinant expression plasmid pET-28a-TAT-HuScFv.

Page 17/19 Figure 5

Purifed TAT-HuScFv. Lane M: marker; lane 1: ultrasonic supernatant induced by TAT-HuScFv expression bacteria; lane 2: Cu2+ 200 mmol imidazole eluted sample; lane 3: purifed TAT-HuScFv.

Page 18/19 Figure 6

Schematic diagram showing peptide fragment 10 decomposed into small peptides (A) and a schematic diagram showing amino acid mutation in polypeptides (B). (A): The green and orange sections are ELISA- positive, and the black circled sections are the amino acid sequences shared by the three polypeptides 5, 6 and 7. (B): The red and yellow sections represent the mutant amino acid sites that are positive for fragment 1 and negative for fragment 2.

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